INTERNATIONAL UNIVERSITY

BIOMEDICAL ENGINEERING DEPARTMENT (BME)

NOVEMBER 19, 2021

BIOLOGY FOR BME

REPORT FOR
LABORATORY

BRADFORD ASSAY – MEASURING

PROTEIN CONCENTRATION USING
ASPECTROMETER
GROUP 4

TRẦN ĐẠO QUANG

NGUYỄN THỊ QUỲNH NGA
NGUYỄN BÌNH PHƯƠNG NHI

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TABLE OF CONTENT
INTRODUCTION4
PURPOSE4
INFORMATION OF MATERIAL4
The coomassie dye4
The mechanism5
Bovine Serum Albumin (BSA) solution6
BSA’s application6
CONTENT8
MATERIAL AND INSTRUMENT8
PBS solution8
Spectrometers8
Spectrophotometer (UV – vis spectrometer)10
Microplate reader11
EXPERIMENTS13
Experiment 1 – Establish a standard absorbance curve for BSA 13
(Bovine Serum Albumin)
Experiment 2 – Determine unknown BSA concentration using Bradford assay14
ANSWERING QUESTIONS15
QUESTION 1 15
QUESTION 221
QUESTION 324
CONCLUSION26
REFERENCES27

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TABLE OF FIGURATIONS
FIGURE 15TABLE II.29
FIGURE 26
FIGURE 38
FIGURE 410
FIGURE 510
FIGURE 612
FIGURE 712
FIGURE 813
FIGURE 915
FIGURE 1016
FIGURE 1116
FIGURE 1217
FIGURE 1317
FIGURE 1418
FIGURE 1518
FIGURE 1619
FIGURE 1719
FIGURE 1820

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I. INTRODUCTION

A. PURPOSE
We learn about Bradford assay, Coomassie dye, bovine serum albumin,
phosphate-buffered saline, spectroscopy, UV-Vis spectroscopy, and microplate
reader: definition, principle, and mechanism. In more detail, we are going to
explore more about measuring protein unknown concentration using a
spectrometer and establishing a standard absorbance curve for Bovine Serum
Albumin (BSA). In terms of Bradford assay, we do learn specifically how to
prepare materials, types of equipment, then record, and statistics the data result
measured. Moreover, we learn to establish a standard absorbance curve for BSA
based on calculations from data numbers. Finally, we will determine the
concentration of proteins in the sample by some experiments that will be
described carefully below.

B. INFORMATION OF MATERIALS
The Bradford assays
a) The Coomassie dye:
The Bradford assay is a rapid and somewhat sensitive technique for
detecting protein quantities. It is based on a change in the maximum
absorbance of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm after
binding to denatured proteins in solution.
Coomassie R-250 (red-tinted) and G-250 (green-tinted) dyes are two
chemical variants of a disulfonated triphenylmethane molecule that is
extensively used as the foundation of stains for protein detection in gel
electrophoresis and Bradford-type assay reagents for protein
quantification.

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Figure 1: Chemical structure

of Coomassie protein assay reagent.

Coomassie Brilliant Blue R-250 and G-250 dyes have the following
characteristics:
Easy detection: creates highly colored complexes with proteins.
High sensitivity: can detect as little as 0.5 g/cm2 of protein in a gel
matrix.
Reversible staining: anion of Coomassie Brilliant Blue formed in the
acidic staining medium electrostatically interacts with the protonated
amino groups of proteins, so the complex is reversible under the
right conditions.
Differentiation of bound and unbound dye: when dissolved in 0.01M
citrate buffer at pH 3.0, exhibits an absorption maximum at 555nm
while the protein-dye complex has a slightly larger peak than the free
dye, having a maximum at 549 nm.
b) The mechanism:
According to the test's concept, the binding of protein molecules to
Coomassie dye under acidic conditions results in a color shift from brown to
blue. Coomassie dye is the chemical form of a disulfonated
triphenylmethane molecule that is extensively employed as the foundation
of stains for protein detection in gel electrophoresis and Bradford-type test
reagents for protein quantification.

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This method detects the presence of the basic amino acid residues
arginine, lysine, and histidine, which contribute to the development of the
protein-dye complex.
c) Bovine Serum Albumin (BSA) solution:
This is also known as Fraction V that is a serum albumin protein isolated
from cows.
In scientific research, it is frequently employed as a protein
concentration standard.

Figure 2: Bovine Serum Albumin

30% solution.

d) BSA’s application:
BSA is used in molecular biology to stabilize certain restriction enzymes
during DNA digestion and to prevent the enzyme from adhering to reaction
tubes, pipet tips, and other vessels.
In many applications, BSA is regarded as a universal blocking reagent
since it has no effect on the functioning of other proteins (enzymes) that do
not require it for stability.
Additionally, BSA is also widely used to assess the number of other
proteins, such as the Bradford Protein Assay, by comparing an unknown
amount of protein to known levels of BSA.

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BSA is used in a variety of biochemical tests, including ELISAs (Enzyme-
Linked Immunosorbent Assays), immunoblots, and immunohistochemistry.
BSA has been frequently employed as a template for the creation of
nanostructures.
And also, BSA is employed because of its stability to boost signal in
assays, lack of influence in many biochemical reactions, and inexpensive
cost, as vast quantities can be easily separated from bovine blood, a
byproduct of the cattle business.

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II. CONTENT

A. MATERIAL AND INSTRUMENTS

1. PBS solution
A typical buffer used in biological research is PBS solution (Phosphate-buffered
saline). It helps to maintain a constant pH. The osmolarity and ion concentrations
of the solutions are equal to those of the human body (isotonic).
PBS has a wide range of uses since it is isotonic and non-toxic to most cells
(e.g. dilution and washing of cell containers). In addition, to disengage stuck
and clumped cells, PBS with EDTA is also utilized. However, divalent metals like
zinc cannot be added since they will precipitate.
When it comes to the transport and storage of RNA viruses like SARS-CoV-2,
PBS has been proved to be a viable alternative to viral transport medium.

Figure 3: Prepare a PBS 1M.

2. Spectrometers
A spectrometer, in the broadest sense, is any instrument that measures the
variation of a physical feature across a given range, i.e. a spectrum.  In an optical

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spectrometer, this could be a change in the absorption and emission of light with
a wavelength, a mass-to-charge ratio spectrum in a mass spectrometer, or a
variation of nuclear resonance frequencies in an NMR
spectrometer. (“Spectrometer Market Size, Share | Analysis & Forecast”).

Types of spectrometer
Name Picture

Optical
spectrometer
(Edinburgh
Instruments FS5
spectrofluorometer).

NMR spectrometer
(Agilent 800 MHz
NMR spectrometer).

Mass spectrometer
(Scion Instruments
GC-MS
spectrometer).

Table II.2: Types of spectrometers.

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3. Spectrophotometer (UV – Vis Spectrometer)
a) Overview:
A spectrophotometer is an instrument that measures the amount of light
absorbed by a sample.
Spectrophotometer techniques are mostly used to measure the
concentration of solutes in solution by measuring the amount of the light that
is absorbed by the solution in a cuvette placed in the spectrophotometer.
Scientist Arnold J. Beckman and his colleagues at the National Technologies
Laboratory (NTL) invented the Beckman DU spectrophotometer in 1940.
b) Working principle:
The spectrophotometer technique is to measure light intensity as a
function of wavelength. It does this by diffracting the light beam into a
spectrum of wavelengths, detecting the intensities with a charge-coupled
device, and displaying the results as a graph on the detector and then on the
display device.
In the spectrophotometer, a prism (or) grating is used to split the incident
beam into different wavelengths.
By suitable mechanisms, waves of specific wavelengths can be manipulated
to fall on the test solution. The range of the wavelengths of the incident light
can be as low as 1 to 2nm.
The spectrophotometer is useful for measuring the absorption spectrum of
a compound, that is, the absorption of light by a solution at each wavelength.

Figure 4: Spectrophotometer. Figure 5: Working principle of

spectrophotometer.

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c) Application:
Detection of concentration of substances.
Detection of impurities.
Structure elucidation of organic compounds.
Monitoring dissolved oxygen content in freshwater and marine ecosystems.
Characterization of proteins.
Detection of functional groups.
Respiratory gas analysis in hospitals.
Molecular weight determination of compounds.
The visible and UV spectrophotometer may be used to identify classes of
compounds in both the pure state and in biological preparations.

4. Microplate reader
a) Overview:
A microplate reader is a laboratory instrument that is used to measure
chemical, biological or physical reactions, properties, and analytes within the
well of a microplate. A microplate consists of small wells in which separated
reactions take place. These reactions convert the presence of an analyte or the
progression of biochemical processes into optical signals. The microplate
reader detects these signals and thus quantifies the parameter of interest.
b) Working principle:
A microplate reader detects light signals produced by samples which have
been pipetted into a microplate. The optical properties of these samples are
the result of a biological, chemical, biochemical, or physical reaction. Different
analytic reactions result in different optical changes used for analysis.
Absorbance, fluorescence intensity and luminescence are the most popular
and most frequently used detection modes in laboratories worldwide.

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Microplate-based measurements detect light signals produced by a sample,
converted by a sample, or transmitted through a sample. The signal is
measured by a detector, usually a photomultiplier tube (PMT). PMTs convert
photons into electricity that is then quantified by the microplate reader. The
output of this process is numbers by which a sample is quantified.

Figure 6: Microplate reader.

Figure 7: Details of microplate reader.

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B. EXPERIMENTS
1. Experiment 1 – Establish a standard absorbance curve for BSA (Bovine
Serum Albumin)
Procedure:
Step 1: Prepare the standard curve
Required volume: 1000 L
BSA concentration
10 20 30 40 50 60
(g/mL)
Volume of stock BSA
5 10 15 20 25 30
(L)

Volume of PBS 1x 995 990 985 980 975 970

Step 2: Add 200 L of BSA [x] and 50 L of Bradford reagent into each well
of the 96-well plate. Mix the solution with micropipette carefully (avoid
creating air bubbles).
Step 3: Incubate at room temperature for 15 minutes after covering the
plate with silver paper.
Step 4: Record the absorbance of BSA using the microplate reader (595
nm).
Step 5: Make a standard curve from the data you have gathered (R2 value is
recommended to be higher than 0.95).
Figure 8: Standard curve.

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2. Experiment 2 - Determine unknown BSA concentration using Bradford
assay
Procedure:
Step 1: Prepare 500 µL of 3 different BSA solutions (unknown
concentration).
Step 2: Add 200 µL of each solution into wells of the 96-well plate.
Step 3: Add 50 µL of Bradford reagent into both of the wells above.
Step 4: Cover the plate in silver paper and incubate at room temperature
for 15 minutes.
Step 5: Use the microplate reader to obtain BSA’s optical density and use
the standard curve to convert that density into real BSA’s concentration.
Step 6: Clean up all the lab instruments and return all of them to the
appropriate places.

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III. ANSWERING QUESTIONS

QUESTION 1:
a) What is Bradford assay? Which reagent does this test use? Which
mechanism does this test rely on?
 The Bradford protein assay is a dye-binding test that uses a dye's
differential color shift in response to varying protein concentrations.
 According to the test's concept, the binding of protein molecules to
Coomassie dye under acidic conditions results in a color shift from brown to
blue. Coomassie dye is the chemical form of a disulfonated
triphenylmethane molecule that is extensively employed as the foundation
of stains for protein detection in gel electrophoresis and Bradford-type test
reagents for protein quantification.

Figure 9: Chemical structure of Coomassie protein assay reagent.

 This
method detects the presence of the basic amino acid residues arginine,
lysine, and histidine, which contribute to the development of the protein-
dye complex.
b) What is spectroscopy?

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 The absorption and emission of light and other radiation by matter is

Figure 10: Dispersion of light by the glass prism. referred

to as
spectroscopy. It entails breaking light (i.e., electromagnetic radiation) into a
spectrum of component wavelengths, just like a prism splits light into a
rainbow of colors.

 Spectroscopy refers to a wide range of techniques that use radiation to

collect information about the structure and characteristics of matter, which
is then used to solve a wide range of analytical issues.
 The wavelengths of each component color are shown in the visible
spectrum. The wavelength varies from 700 nm (dark red) - 400 nm (violet).
Additionally, UV (Ultraviolet) is a kind of electromagnetic radiation with a
wavelength that ranges from 10 nm - 400 nm, which is shorter than the
wavelength of visible light.
 The interaction of radiation with matter is determined by both radiation
characteristics and structural components of the materials involved. This

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interaction between matter and radiation produces a wide range of
spectra.

c) Inside the Labs of BME, there is a UV-VIS Spectrophotometer and a

Microplate Reader. Describe how these machines work and identify the
differences.
UV-VIS Spectrophotometer:
 UV-Vis Spectroscopy (also known as Spectrophotometry) is a quantitative
technique for determining how much light a chemical compound absorbs.

Figure 11: Electromagnetic spectrum.

Figure 12: UV – Vis spectrophotometer.

Figure 13: The optical configuration of a Modern UV-Vis Spectrophotometer.

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 Spectrophotometry is the quantitative measurement of a material's
absorption or transmission or reflection as a function of wavelength.
Despite being UV-Vis, the wavelength range that is commonly employed
runs from 190 nm - 1100 nm in the near-infrared.
 We may calculate the amount or concentration of a given chemical
compound by measuring the number of photons (i.e. Light intensity) that
reach the detector using a spectrophotometer and absorption or
transmission measurements. The higher the quantity of a recognized
component, the more a material absorbs light at a certain wavelength.
Microplate Reader:
 A microplate reader is a laboratory tool
used to monitor chemical, biological, or
physical reactions, characteristics, and
analytes within a microplate well. A
microplate is made up of tiny wells in which
different reactions occur. And, the presence
of an analyte or the advancement of
biological processes is converted into optical
Figure 14: Microplate reader.
signals by these reactions. Especially, these
signals are detected by the microplate reader, which quantifies the
parameter of interest.
 Microplate-based measurements detect light signals produced by samples
that have been pipetted into a microplate, converted by a sample, or
transmitted through a sample. The signal is measured by a detector, usually
a photomultiplier tube (PMT). They convert photons into electricity that is

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then quantified by the microplate reader. Then the output of this process is
numbered by which a sample is quantified.

Figure 15: A microplate reader has 96 wells.

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 Furthermore, depending on the nature of the optical signal changes during

a reaction and consequently, on the detection mode, samples may need to
be excited by light at specific wavelengths. This light is usually provided by a
broadband xenon flash lamp. In order to allow excitation of the sample only
by specific wavelengths, the light produced by the lamp is selected by a
specific excitation filter or monochromator. To increase sensitivity and
specificity, filters or monochromators are equally employed on the
emission or detection side. These are usually placed between the sample
and the detector.

Figure 16: Depicts the distinctions between

the UV-Vis Spectrophotometer system and
the Microplate Reader system.

There are several

differences between a microplate reader and a UV-Vis spectrophotometer:
 First difference between is the shape of the cuvette of (a) a cuvette used
in the spectrophotometer and (b) a microtiter plate used in the
microtiter plate reader system. While indicating the optical intensities
before and after detection according to an arrow direction, at I 0 and I,
respectively.
 Second, the sample size of the spectrophotometer works with samples
held in a cuvette, measuring about 1 µL at a time. This allows for very
accurate readings and a high dynamic range, saving on test sample
material costs but time-consuming research. In contrast, a plate reader
works with word counts of 96 wells (most common) or larger. This
means that in less time, the microplate reader returns multiple sample
results.

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 Third, the way how the light beams are delivered:
In the spectrophotometer, the beams travel horizontally across the
cuvette, and this route length is commonly set at 1 cm. This contributes
to the increased degree of accuracy provided by the

Figure 17: Depict how the light beams are delivered in the UV-Vis Spectrophotometer.

spectrophotometer. On the other hand, this high level of detection

might produce artifacts and trace components that can confound
measurements.

 In a microplate reader, light is transported vertically through the wells.

However, this orientation introduces factors that must be considered. And
also, pipetting precision and consistency are critical since the volume within
the well dictates the path length. In addition, shaking settings on microplate
readers are frequently used to assist eliminate any bubbles from the wells
that might affect the curvature of the meniscus. Regardless of the variables
that may develop, the majority of plate readers on the market have
strategies to adjust and offer consistent and trustworthy readings.

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QUESTION 2:
Material and equipment:
TABLE OF QUESTION 2
Material Equipment
Micropipettes.

Bradford reagent.

1 Eppendorf tubes rack.

BSA 1mg/mL.

10 Eppendorf tubes.

PBS 1x solution.

2 96- well plate.

10 mg Coomassie dye (Brilliant blue

G250).

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Microplate reader.

10 mL Methanol (absolute).

Pipette.

20mL H3PO4.

Beaker.

10mL Distilled water.

Analytical balance.

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Procedure:
Step 1: Prepare the 40mL of Bradford reagent:
 Use an analytical balance to obtain approximately 10mg of
Coomassie dye (Brilliant blue G250). Besides, use Pipette 10 mL
methanol into a beaker, dissolve Coomassie dye (Brilliant blue
G250) with methanol.
 Add 10ml of distilled water to the solution in the beaker.
 After that, we should add 20mL of H3PO4.
Step 2: Prepare the standard curve.

Required volume: 1000 L

BSA concentration
10 20 30 40 50 60
(g/mL)

Volume of stock BSA

10 20 30 40 50 60
(L)

Volume of PBS 1x 990 980 970 960 950 940

Step 3: Fill each well of the 96-well plate with 200  l of BSA [x] and 40ml of
Bradford reagent. Carefully mix the solution with a micropipette.
Step 4: Incubate at room temperature for 15 minutes after covering the
plate with silver paper.
Step 5: Use the microplate reader to record BSA’s absorbance (595 nm).
Step 6: Create a standard curve using the data you have gathered (R2 value
is recommended to be higher than 0.95).

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QUESTION 3:
a) We calculate the average absorbance of each sample from the table below:

Then we have the results of each sample in several protein concentrations

STANDARD CURVE
in this table below: Protein
concentration Absorbance
[µg/mL]
10 0.147
20 0.2747
30 0.4443
60 0.8706
80 1.1804
100 1.4604
120 1.764
From this table, we can draw the standard curve by excel and then we can
get the function:

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So: the function is y = 0.0147x – 0.0068
R2 = 0.9998
b) From the function, we have: FUNCTION
y: absorbance Slope 0.0147
x: protein concentration Intercept 0.0068

From the given absorbance and the function with slope and intercept, we
can calculate the protein concentrations and the table below shows the
rounded results of protein concentration of each unknown sample:
Unknow sample Absorbance Concentration [µg/mL]
Sample 1 0.39 27
Sample 2 0.243 17
Sample 3 0.949 65.02
Sample 4 0.493 (1:50) 1700

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IV. CONCLUSION

In summary, the Bradford protein test is a spectroscopic protein analysis

process that can be done quickly. When determining the concentration of
protein in a solution, it has a high degree of accuracy. Because of its
apparent simplicity and sensitivity to a wide range of proteins, the Bradford
assay is popular and frequently used. To calculate the absorbance of
standards and unknown samples, spectrophotometry is used. As a result, if
a container (or, more officially, a cuvette) of opaque liquid is placed
between the spectrometer and the photometer, the photometer reading
will vary depending on the amount of light that can flow through the
cuvette, and the absorbance level can be recorded. We were able to
measure the absorbance of the standards, plot them, and utilize them as a
basis for determining the identities of unknown chemicals using these
approaches.

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REFERENCES

1. What is Spectroscopy? (news-medical.net)

2. Coomassie Brilliant Blue G-250 Dye (thermofisher.com)
3. Bradford Protein Assay - an overview | ScienceDirect Topics
4. Ultraviolet (UV) Radiation | UCAR Center for Science Education
5. UV Vis Spectroscopy | UV Vis Spectroscopy Applications | Edinburgh Instruments
(edinst.com)
6. Microplate Reader | Plate Reader - BMG LABTECH
7. Microplate
Rhttps://www.wikihow.com/Make-Phosphate-Buffered-Saline#/Image:Make-
Phosphate-Buffered-Saline-Step-4.jpg
8. https://www.edinst.com/blog/what-is-a-spectrometer/
9. Reader VS Spectrophotometer: What’s the difference? - The Lab World Group
10. https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/
11. https://www.bmglabtech.com/microplate-reader/

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POINT FEEDBACK

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BIOLOGY FOR BME – LABORATORY

BRADFORD ASSAY - MEASURING PROTEIN
CONCENTRATION USING ASPECTROMETER
GROUP 4
THE END

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